Influence Of The Expression Of Epidermal Growth Factor Receptor To Radiosensitivity In Nasopharyngeal Carcinoma Cell

Nasopharyngeal carcinoma (NPC) is an endemic cancer with high incidence inSoutheast Asia and Southern china. Although great progress in the investigation ofNPC, such as radiotherapy and imaging diagnosis and so on, have been made in thelate 90s,the cure rate of NPC has not been improved yet and 5 year survival rate stillremained around 50-60％.Radiotherapy is the most important treatment method for NPC. Treatmentfailure is mainly because of local relapse and metastasis, which decreases thesurvival rate obviously. Radiotherapy is a regional treatment.A better local curativeeffect of radiotherapy not only increases local contral rate, but also affects survivalrate and metastasis. Oncologists have been investigating the approach to increase thecurative effect of radiotherapy and radiation sensitivity.The radiosensitivity of tumor is a very complicated problem and the impactfactor involves in many aspects. It is decided by organize source, differentiationdegree, pathological type and the bed of tumor, anemia and partial infection, lifeindex…etc. The messures to modify the radiotherapy effects before are as follows: 1.anoxic cell radiosensitizer. 2. radiosensitizer of tumor cell, halogen as principleamong them, but are't used widely. 3. to change oxygen content of tumor by inhaling different density of oxygen 4.radiotherapy combined with chemotherapy,but thismight increase the side effects of normal tissue 5.radiotherapy combined with othermeasures such as thermotherapy,photodynamic therapy and so on.Epidermal growth factor receptor (EGFR) is a sort of receptor tyrosine kinase(RTK). It was first discovered by Cohen and his colleagues in 1982. Thetransmembrane protein receptor with tyrosine kinase activity in cellular membraneplays an important role in cell signal transduction.It breakdowns a series of signalcascade reactions that induce cell proliferation and differentiation. Epidermal growthfactor receptor family is a very important receptor signal systerm which involves inthe cell proliferation and tumorigenesis. Many oncogenes can encode EGFR or thesimilar protein that lead an important function in the growth of most of tumororiginated from epithelium tissue of mankind. The overexpression of EGFR familyalways causes the sensitivity changes of radiotherapy and chemotherapy of tumor.We can restrain the growth of tumor and change the sensitivity of radiotherapy,chemotherapy and hormone therapy through inhibiting the expression of EGFR bymolecular targeted therapy or gene technology. Epidermal growth factor receptorfamily includes four members: EGFR,HER-2/P185erbB2,HER-4/P180erbB4 andHER-3/P160erbB3.EGFR (HER1,erbB1) gene,localizing in chromosome 7q21, encodes aglycoprotein receptor with tyrosine kinase activity. The phosphorylation of itself afterintegrating with corresponding ligand results in a trial of signal cascade amplificationand transmitting. At last, it affects the expression of intranuclear genes and inducescell proliferation and differentiation. EGFR are widely distributed on the surface ofendothelial cell of normal mammal, 5×10~4～10×10~4 receptors in every cell average.There are six kind of ligands of EGFR at present:EGF, TGF-a, amphiregulin,BTC,HB-EGF and EPR. BTC,HB-EGF and EPR are the co-ligand oferbB receptor family,but EGF, TGF-a and amphiregulin just bind on EGFR.ActivitedEGFR can activate several downstream signal transduction pathway. After Hendlerhad found the overexpression of EGFR of non small cell lung cancer at the firsttime,the overexpression or amplification was detected in several solid tumor asbreast,colorectal, ovarian, head and neck cancer, cerebroma,carcinoma of bladderand renal carcinoma. The overexpression of EGFR of tumor is an important index ofunfavourable prognosis and radiation resistance. EGFR is associated with invasionand metastasis of tumor also. In addition, EGFR can affect the reconstitution, shift,sticking of cystoskeleton and protease activity.Can radiosensitivity of tumor be changed by blocking up or restraining theexpression of EGFR? It is an ideal targeted anticancer therapy method to restrainEGFR. In recent years, RNA interference(RNAi) develops very quickly as apragmatic molecular biology technology besides molecular targeted therapy. RNAican inhibit gene expression specificly in high performance. In practically, it is morevaluable than other technologis of intergenic suppression. The transient or stabilegene silencing mediated by siRNA has a high specificity and no side effect. There isno report about the changes of radiosensitivity of nasopharyngeal carcinoma cell afterEGFR was restrained with RNAi up to now in literatures.Our studies included:Part One: The expression of epidermal growth factor receptorfamily of nasopharyngeal carcinoma CNE1 and CNE2 cell linesObjiective: To detect the expression of EGFR, HER-2, HER-3 and HER-4 ofnasopharyngeal carcinoma CNE1 and CNE2 cell lines. Methods: 1.The expression ofEGFR, HER-2, HER-3 and HER-4 of nasopharyngeal carcinoma CNE1 and CNE2 lines were tested by immunohistochemistry(IHC),Western blot and RT-PCR. 2. Therelation of radiation dose and survival rate of cell was study by colony formationexperiment to prepare the conditions of the irradiation. Result: 1. The expressionof EGFR of CNE1 and CNE2 protien was positive, HER-2, HER-3 and HER-4negative detected by IHC and Western blot. 2. The expression of EGFRmRNA ofCNE1 and CNE2 was positive, HER-2, HER-3 and HER-4 negative detected byRT-PCR. 3. After CNE1 and CNE2 were irradiated 2Gy with X-ray, the survivalrate is about 50% on the 12th day; after were irradiated 4Gy, 22.93% and 3.67%respectively. Conclusion: The expression of EGFR of CNE1 and CNE2 was positive,HER-2, HER-3 and HER-4 negative.Part Two: To construct, transfect and identity the EGFRSiRNAexpression vectorObjective: To construct the EGFRSiRNA expression vector and complete thetransfection and identity, and detect the expression of EGFR after tranfection.Methods: The nonsense and vacant vector were transfected as control besides EGFRinterference plasmid. DNA sequencing was deployed. Expression of EGFRmRNAwas detected before and after transfection by real-time fluorescence quantitive PCR.Expression of EGFR protein after having tansfected different vector was detected byWestern blotting. Result: 1. EGFR interference vector was constructed successfully.2. Expression of EGFRmRNA of CNE1 and CNE2 was detected by real-timefluorescence quantitive PCR: expression of EGFRmRNA of CNE1 and CNE2 cellswas decreased in transfecting EGFR interference vector group in contrast to nonsenseand vacant vector group. In statistics analysis, the difference of EGFRmRNAexpression of CNE1 cells between transfecting EGFR interference vector group andnonsense group was significant(P=0.000), so was between EGFR interference vector group and vacant vector group(P=0.000). In transfecting EGFR interference vectorgroup, EGFRmRNA was decreaced by degree at 0,12,24,48,60,72 hour aftertransefection, the difference was significant between before transfecting and 48,60,72hour after transfecting(P=0.048,0.011,0.013). In statistics analysis, the difference ofEGFRmRNA expression of CNE2 cells between transfecting EGFR interferencevector group and nonsense group was significant(P=0.000), so was between EGFRinterference vector group and vacant vector group (P=0.000). In transfecting EGFRinterference vector group, EGFRmRNA was decreaced by degree at 0,12,24,48,60,72hour after transefection, the difference was significant between before transfectingand12,24, 48,60,72 hour after transfecting (P=0.004,0.003,0.000,0.000,0.000).3. Expression of EGFR protein was detected by Western blotting: expression ofEGFR protein of CNE1 and CNE2 cells was decreased after transfecting EGFRinterference vector compared with transfectingβ-actin, vacant and nonsense vector.Conclusion: 1. SiRNA expression plasmid was constructed and transfectedsucceesfully. 2. Expression of EGFRmRNA of CNE1 and CNE2 cells was decreasedafter transfecting EGFR interference vector. 3. Expression of EGFR protein of CNE1and CNE2 cells was decreased after transfecting EGFR interference vector.Part Three: The change of radiosensitivity of CNE1 and CNE2 cellsafter EGFR interferenceObjective: To study the change of radiosensitivity of CNE1 and CNE2 cellsafter interference. Methods: 1. The apoptosis of CNE1 and CNE2 cell before andafter interfering was observed by TUNEL, DNA LADDER, electron microscope andDAPI after irradiated by X-ray. 2. The ratio of apoptosis and necrosis of CNE1 andCNE2 cell was detected by Annexin V/PI, and was compared before and afterinterfering vector after irradiation. 3. The proliferation of cells irradiated was detected by MTT experiment, and was compared before and after interfering. Result: 1.Apoptosis can be found after irradiation or interference by TUNEL, DNA LADDER,electron microscope and DAPI. 2. Detected by Annexin V/PI, apoptosis and necrosisof CNE1 and CNE2 cells irradiated increased befoe and after interference; theproportion of apoptosis was more than necrosis. In statistics analysis, the difference ofapoptosis ratio of CNE1 cells treated by irradiation between before and afterinterference was significant(P=0.008), and it changed following the timesignificantly(P＜0.05); the necrosis ratio of CNE1 cells treated by irradiation betweenbefore and after interference was not significantly different(P=0.082), but it changedfollowing the time significantly(P＜0.05). The difference of apoptosis ratio of CNE2cells treated by irradiation between before and after interference was significant(P=0.020), and it changed following the time significantly (P＜0.05); the necrosis ratioof CNE2 cells irradiated between before and after interference was significantlydifferent(P＜0.05), it changed following the time significantly(P＜0.05) except between24h and 48h after irradiation(P=0.170). 3. Detected by MTT experiment: proliferationof CNE1 and CNE2 cells irradiated was decreased after interference. In statisticsanalysis, the proliferation difference of CNE1 cells irradiated was significant betweenbefore and after interference(P＜0.001); before interference: the difference betweenbefore irradiation and at 30,36,48,60,84,96 hour after irradiation was significant(P=0.037,0.002,0.017,0.003,0.006,0.013); after interference, the differencebetween before irradiation and at 24,36,48,60,72,84,96 hour after irradiation wassignificant (P=0.037,0.037,0.002,0.001,0.001,0.001,0.003). The proliferationdifference of CNE2 cells irradiated was significant between before and afterinterference(P＜0.001); before interference: the difference between before irradiationand at 36,48,60,84,96 hour after irradiationis was significant (P=0.002,0.031,0.005,0.010,0.033); after interference, the difference between before irradiation and at 36,48,60,72,84,96 hour after irradiation was significant(P=0.047,0.002,0.001,0.001,0.001,0.003). Conclusion: 1. Apoptosis of CNE1 and CNE2 cells could befound after irradiation by TUNEL, DNA LADDER, electron microscope and DAPI. 2.The ratio of apoptosis and necrosis of CNE1 and CNE2 cells irradiated was increasedafter interference, the proportion of apoptosis was higher than necrosis. 3. Theproliferation of CNE1 and CNE2 cells treated by irradiation was decreased afterinterference. The proliferation of tumor cells irradiated was restrained after theexpression of EGFR had been decreased by RNA interference. The radiosensitivity ofNPC CNE1 and CNE2 cells was increased after the expression of EGFR had beendecreased by RNA interference.